(1) Field of the Invention
The present invention relates to a monomer protein having an amino acid sequence belonging to TGF-β superfamily, of which cysteine related to a dimer formation of a protein has been replaced with another amino acid. Moreover, the present invention relates to a method for preparing said monomer protein in a large amount and with a high purity by using Escherichia coli transformed with a plasmid containing a DNA sequence that can express said monomer protein. Furthermore, the present invention relates to an agent containing said monomer protein for preventing and treating a disease affecting bone and/or cartilage.
(2) Description of the Related Art
Currently, there are known estrogen, calcitonin, vitamin D3, its derivatives and derivatives of bisphosphonic acid as preventive or therapeutic agents for bone diseases. Recently, it has been reported that a bone morphogenetic activity is found in a series of a bone morphogenetic protein (hereinafter referred to as “BMP”) belonging to TGF-β superfamily, from BMP-2 to BMP-14.
Moreover, it has been reported that a protein named GDF-5 or human MP52 has a bone morphogenetic activity (WO93/16099, WO95/04819, WO94/15949 and Nature Vol. 368, 1994, p. 639-643). It is considered that mature human MP52 is a protein having 120 amino acid residues starting with alanine at an N-terminal, and its amino acid sequence has been described in these patent applications.
These proteins exist as a homodimer having a single disulfide bond in nature. On the contrary, the manufacture of their recombinant protein is carried out using their homodimers or heterodimers to yield a protein showing the activity. For example, human MP52 has been reported in the publication of unexamined application, JP 031098/97.
Meanwhile, there are two types named type I receptor and type II receptor in the receptors of TGF-β superfamily. Intercellular signal transmission via receptors of TGF-β superfamily containing these bone morphogenetic proteins (dimers) requires simultaneous combination of these proteins to both type I and type II receptors, and it is considered that a polymer is formed by gathering of two or more dimers to do intercellular signal transmission (Bone, Vol. 19, 1996, p. 569-574). It has been considered that for polymer formation it is important that the protein should be a dimer. The activity in a monomer has not yet been found. Moreover, preparation for these monomer recombinants has not yet been carried out.